Summary: Chronic infection with the hepatitis C virus (HCV) is a major cause of cirrhosis, decompensated liver disease and hepatocellular carcinoma (HCC) worldwide. Globally there are an estimated 72 million persons with HCV. In the United States (U.S.), 1% of the general population or 2.7 million persons have chronic infection based on the detection of HCV RNA in serum. In 2014, chronic HCV infection was the leading infectious cause of death in the U.S. These figures underscore the magnitude and impact that chronic HCV infection has on global and US public health. The natural history of chronic HCV infection has been difficult to study. The protracted and silent course of infection, the absence of large cohorts of persons known to be infected, and the wide variability in outcome are major obstacles to natural history studies. Five to twenty-five percent of HCV-infected persons will develop cirrhosis over a 25-30 year period but some patients remain asymptomatic, without significant liver disease for many decades if not for life. Knowledge of the rate of progression among individuals who have not developed cirrhosis is unknown. An equally important and related issue is the clinical assessment of disease severity. Unfortunately, there are no good laboratory markers of disease severity and liver biopsy, the accepted gold standard for assessing disease severity is imperfect. Non-invasive methods to assess disease severity are highly desirable for the clinicians diagnostic toolbox. The optimal treatment for chronic HCV infection is undergoing a paradigm change with the development of direct acting antiviral (DAA) agents. For most genotypes response rates to treatment now approach 90-95%. The long-term outcome of patients who are treated with DAAs remains to be determined. In particular, what is the incidence of hepatocellular carcinoma and how quickly does liver fibrosis/cirrhosis regress are important questions that remain to be answered. Hypotheses/problems addressed: 1) Define the host, viral and environmental factors that determine the natural history and outcome of HCV infection. To address this problem, we have submitted a new protocol to prospectively follow a cohort of 350 subjects successfully treated with direct acting antiviral agents to define the long-term outcome of patients after eradication of hepatitis C virus. The two primary aims of the study are to determine prospectively the rate of clinical outcomes (ascites, spontaneous bacterial peritonitis, hepatic encephalopathy, variceal hemorrhage), hepatocellular carcinoma, liver-related mortality and all-cause mortality in treatment recovered hepatitis C and to determine the proportion and rates of fibrosis regression in subjects after successful therapy of chronic hepatitis C. A major focus of the study will be to develop novel biomarkers and genetic predictors of liver-related complications including hepatocellular carcinoma. Cases of HBV reactivation among patients with chronic HCV infection while undergoing therapy with direct acting antiviral agents have been reported in the literature. In collaboration with the University of Toronto, we are retrospectively assessing the rate and features of HBV reactivation in a group of 90 patients. 2) Assessment of cardiovascular risk after HCV eradication Hepatitis C virus (HCV) exploits the host lipoprotein pathway for its lifecycle. Consequently, patients with chronic HCV have hypolipidemic profile. This observation suggests that sustained eradication of HCV might lead to dyslipidemia and increase risk for cardiovascular disease. Serial lipid panels and NMR-based lipoprotein profiles were assessed at multiple timepoints from fasting chronic HCV patients participating in a study evaluating sofosbuvir/velpatasvir for 12 weeks were evaluated. We have previously demonstrated a marked rise in all lipoprotein parameters within the first 7 days of antiviral treatment associated with viral clearance. The increase in LDL and more importantly small LDL (the most atherogenic particle) persisted 24 weeks after treatment was stopped. As a follow-up we have shown by qPCR using paired liver biopsy samples that rapid clerance of HCV from hepatocytes appears to upregulate cholesterol, lipoprotein and fatty acid biosynthesis through induction of key regulators of lipid and lipoprotein metabolism. These results suggest that patients should be evaluated for cardiovascular risk post-SVR and assessed for lipid lowering therapy. 3. Elucidate the viral pathogenesis of chronic HCV infection and mechanisms of action of anti-viral therapy In association with a clinical study using asunaprevir and daclatasvir (direct acting antiviralsDAAs) we sought to answer three critical questions surrounding therapy of chronic hepatitis C: (1) what happens to the expression of interferon stimulated genes (ISGs) following suppression of viral replication in treatment responders versus non-responders (2) can interferon responsiveness be restored in non-responders after suppression of viral replication and (3) why do patients with cirrhosis respond less compared to those without cirrhosis. To address the first question, we compared the relative expression level of ISGs from paired liver biopsy tissue before and after suppression of viremia with potent DAAs. We demonstrated that the level of ISGs decline rapidly, within 2 weeks following suppression of viral replication and this decline was highly correlated with suppression of viremia. Interestingly, subjects who responded had higher baseline expression of ISGs and NK cell activation compared to patients who experienced virological breakthrough (failed therapy). These results suggest that innate immunity may be required for clearance of HCV during DAA therapy. Because miRNAs are known to be important for the viral life cycle, we then asked whether miRNAs were important for viral clearance during therapy with DAAs. Using an unbiased correlation analysis of the hepatic expression data we identified 14 miRNAs that were significantly negatively correlated with ISGs between paired biopsies. 13/14 miRNAs had known ISGs as targets. Interestingly, 9/14 miRNAs were significantly lower at baseline in responders compared to non-responders and increased at week 2 or 4 of treatment suggesting regulation by HCV. These novel results suggest that miRNAs may directly and indirectly regulate innate immunity, ISGs production and contribute to viral clearance during DAA therapy and regulate the immune response to the virus. To further demonstrate that the identified miRNAs regulate ISGs and viral replication we overexpressed/inhibited 3 candidate miRNAs in HUH-7 cells, a hepatoma cell line, and measured levels of predicted target ISGs using qPCR. We confirmed that miRNAs do affect ISG expression. Next, we infected the HUH-7 cells with HCV and demonstrated that the miRNAs affect HCV replication through an effect on ISGs. Taken together, this work suggests that viral replication is the driver for ISG induction in the liver. MiRNAs have a dual role to both impact viral replication and to regulate the innate immune response to the viral infection.